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Regulation of Phosphatidylinositol 3′-Kinase by Tyrosyl Phosphoproteins

FULL ACTIVATION REQUIRES OCCUPANCY OF BOTH SH2 DOMAINS IN THE 85-kDa REGULATORY SUBUNIT (∗)
  • Tamara Rordorf-Nikolic
    Affiliations
    (1) Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Debra J. Van Horn
    Affiliations
    (1) Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Daxin Chen
    Affiliations
    (1) Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Morris F. White
    Affiliations
    (2) Joslin Diabetes Center, Research Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215
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  • Jonathan M. Backer
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2153; Fax: 718-829-8705
    Affiliations
    (1) Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Author Footnotes
    ∗ This work was supported by National Institutes of Health Grant DK-44541 and grants from the Juvenile Diabetes Foundation and the Alexandrine and Alexander L. Sinsheimer Fund (to J. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:February 24, 1995DOI:https://doi.org/10.1074/jbc.270.8.3662
      Phosphatidylinositol 3′-kinase (PI 3′-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3′-kinase in vitro. However, activation by the mono-phosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3′-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3′-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3′-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3′-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3′-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3′-kinase by tyrosyl-phosphorylated proteins requires occupancy of both SH2 domains in p85.

      INTRODUCTION

      Phosphatidylinositol 3′-kinase (PI 3′-kinase)
      The abbreviations used are: PI 3′-kinase
      phosphatidylinositol 3′-kinase
      RCM
      reduction and carboxymethylation of cysteine residues
      IRS-1
      insulin receptor substrate-1
      P-IRS-1
      phosphorylated IRS-1.
      is a lipid kinase that has been implicated in the regulation of cell growth by growth factor receptors and oncogene products(
      • Parker P.J.
      • Waterfield M.D.
      ). PI 3′-kinase phosphorylates phosphatidylinositol at the D-3 position of the inositol ring(
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ), and stimulation of cells with mitogens such as platelet-derived growth factor or transformation of cells with polyoma middle T antigen leads to increases in the levels of the lipid products PI 3,4-P2 and PI 3,4,5-P3(
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Ling L.E.
      • Drucker B.J.
      • Cantley L.C.
      • Roberts T.M.
      ). The function of these lipids has not yet been determined, but their low abundance and rapid appearance in mitogen-stimulated cells suggests that they may serve as intracellular second messengers.
      PI 3′-kinase is a heterodimer composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110)(
      • Parker P.J.
      • Waterfield M.D.
      ). During activation of PI 3′-kinase by tyrosine kinase receptors such as the platelet-derived growth factor or colony stimulating factor-1 receptors, the SH2 domains of p85 bind directly to specific phosphorylated YXXM motifs present in the cytoplasmic domains of the receptors (reviewed in (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). In the case of the insulin receptor, PI 3′-kinase binds to phosphorylated YXXM motifs in the substrate IRS-1 to a greater extent than to the receptor itself(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Backer J.M.
      • Myers Jr., M.G.
      • Sun X.-J.
      • Chin D.J.
      • Shoelson S.E.
      • Miralpeix M.
      • White M.F.
      ). The binding of receptors or substrates containing phosphorylated YXXM motifs activates the lipid kinase in vitro and in intact cells(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ). Activation of PI 3′-kinase in vitro can be mimicked by synthetic phosphopeptides that contain the YXXM motif, and this peptide-mediated activation correlates with conformational changes in the p85 subunit(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Panayotou G.
      • Bax B.
      • Gout I.
      • Federwisch M.
      • Wroblowski B.
      • Dhand R.
      • Fry M.J.
      • Blundell T.L.
      • Wollmer A.
      • Waterfield M.D.
      ). Thus, regulation of PI 3′-kinase catalytic subunit p110 appears to require conformation changes in the regulatory subunit p85, which are driven by SH2 domain binding to phosphorylated YXXM motifs. Recently, several additional mechanisms of activation have been described, including the binding of Src family SH3 domains to p85 (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ) and the binding of p21ras to p110(
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). In contrast, PI 3′-kinase activity is reduced 80% by phosphorylation of Ser608 in p85 and can be restored by treatment with phosphatase 2A(
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.-M.
      • Schaffhausen B.
      • Cantley L.C.
      ).
      In our previous study(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ), we found that activation by mono-phosphorylated YXXM peptides is significantly less potent than activation by the multiply phosphorylated IRS-1; based on these data, we proposed that full activation of PI 3′-kinase requires occupancy of both SH2 domains. This hypothesis has been supported by studies showing that bis-phosphopeptides exhibit enhanced activation of PI 3′-kinase relative to mono-phosphopeptides(
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Herbst J.J.
      • Andrews G.
      • Contillo L.
      • Lamphere L.
      • Gardner J.
      • Lienhard G.E.
      • Gibbs E.M.
      ). In the present study, we have directly tested our hypothesis using recombinant PI 3′-kinase containing disabling mutations in the N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domain reduces phosphopeptide binding to p85 and reduces maximal PI 3′-kinase activation by 50%. Mutation of both SH2 domains abolishes both binding and activation. These data demonstrate that full activation of PI 3′-kinase requires occupancy of both SH2 domains in the p85 regulatory subunit.

      DISCUSSION

      SH2 domains have been implicated in the regulation of an increasing number of proteins involved in signal transduction by growth factors and oncogene products(
      • Pawson T.
      • Schlessinger J.
      ). For the PI 3′-kinase, the binding of its SH2 domains to phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs can directly regulate its activity(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ). We and others (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Herbst J.J.
      • Andrews G.
      • Contillo L.
      • Lamphere L.
      • Gardner J.
      • Lienhard G.E.
      • Gibbs E.M.
      ) have shown that peptides or proteins containing multiple phosphotyrosine residues bind and activate PI 3′-kinase with enhanced potency relative to mono-phosphopeptides. However, these studies cannot differentiate between a mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. In the present study, we show that point mutations in either the N- or C-terminal SH2 domains of p85 inhibit phosphopeptide binding to p85 and reduce the activation of PI 3′-kinase by IRS-1 by 50%. These data are the first direct demonstration that full PI 3′-kinase activation by tyrosyl phosphoproteins requires occupancy of both SH2 domains.
      As we have previously noted, a number of the receptors that activate PI 3′-kinase have pairs of tyrosine phosphorylation sites that may be involved in the bivalent binding of p85 SH2 domains(
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ). Thus, the kinase insert region of the platelet-derived growth factor β-receptor contains two tyrosine phosphorylation sites, both of which are important for PI 3′-kinase binding(
      • Kazlauskas A.
      • Cooper J.A.
      ,
      • Kazlauskas A.
      • Cooper J.A.
      ,
      • Kashishian A.
      • Kazlauskas A.
      • Cooper J.A.
      ,
      • Yu J.C.
      • Heidaran M.A.
      • Pierce J.H.
      • Gutkind J.S.
      • Lombardi D.
      • Ruggiero M.
      • Aaronson S.A.
      ). Recent studies(
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Reedijk M.
      • Liu X.
      • van der Geer P.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ) have also suggested that two phosphotyrosine residues are required for activation of PI 3′-kinase by the hepatocyte growth factor receptor and polyoma middle T antigen. Mutation of Tyr721 but not Tyr697 or Tyr706 within the colony stimulating factor-1 receptor kinase insert domain blocks PI 3′-kinase binding (
      • Reedijk M.
      • Liu X.
      • van der Geer P.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ); however, activation of PI 3′-kinase was not examined and may require an additional phosphotyrosine residue. In contrast, in vitro activation of PI 3′-kinase by a peptide containing the motif Y1322THM from the insulin receptor C terminus is unaffected by the phosphorylation of the neighboring Tyr1316(
      • Van Horn D.J.
      • Myers Jr., M.G.
      • Backer J.M.
      ). The ability of a second phosphotyrosine residue to augment the activation by a phosphorylated YXXM motif presumably depends on the primary sequence surrounding the second phosphotyrosine residue or perhaps the distance between the residues. In this regard, the optimal spacing between phosphotyrosine residues for PI 3′-kinase activation has not been determined. The two phosphotyrosine residues in the IRS-1-derived peptide examined here are 20 amino acids apart, whereas the phosphotyrosine residues in activating peptides derived from polyoma middle T and the hepatocyte growth factor receptor are 7 residues apart; those in the insulin receptor bis-phosphopeptide, which does not show enhanced activation relative to mono-phosphopeptides, are only 6 residues apart.
      Little is known about the native tertiary structure of p85, but these observations suggest that the phosphopeptide binding sites of the p85 SH2 domains must lie quite close to each other. The demonstration that p110 binds to a region between the two SH2 domains of p85(
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ,
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Hu P.
      • Schlessinger J.
      ) suggests an intriguing model in which occupancy of both SH2 domains causes a conformational change in the intervening region and activates p110. In contrast, a recent report showed that a construct containing either SH2 domain plus the intervening region could bind to the p110 catalytic subunit and mediate phosphopeptide-stimulated activation (
      • Holt K.H.
      • Olson A.L.
      • Moye-Rowley W.S.
      • Pessin J.E.
      ); it is not clear how the structure of this truncated construct relates to that of the intact p85 molecule. The resolution of these questions must await a better structural characterization of the intact regulatory and catalytic subunits of PI 3′-kinase.

      Acknowledgments

      We thank Dr. Charles Dahl (Harvard Medical School) for his assistance with phosphopeptide synthesis, Dr. James C. Kauer for his gift of benzoylphenylalanine, Dr. Michael Waterfield for providing the p85 and p110 constructs, and Drs. Joseph Schlessinger and Patrick Hu for providing the p85 glutathione S-transferase fusion proteins and the anti-p85 antibody used in the activation experiments.

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